Weight loss methods and techniques have involved some form of reducing caloric consumption or increasing exercise output to result in utilization of fat stores (e.g., triglycerides) as an energy source. Reducing consumption-type therapies have included various sympathomimetic agents, such as amphetamines and derivatives to increase metabolic rate while reducing the individuals appetite in an effort to reduce caloric consumption.
Numerous attempts have been made, with varying degrees of success and varying degrees of risk to the individual, to reduce body weights of an individual through reduction of fat stores. One general problem with most weight loss programs or therapies is that starving or reducing caloric intake results in metabolism of both fat stores and muscle mass as energy sources for body metabolism. Therefore, one goal of weight loss therapies is to be able to reduce body fat stores while not reducing muscle mass.
There are two types of mammalian fat cells, white fat cells and brown fat cells. Brown fat cells are characterized by the presence of numerous mitochondria that have unique metabolic capacity. Brown fat mitochondria completely catabolize or oxidize fatty acids as a fuel substrate without utilizing the energy source. More specifically, brown fat mitochondria oxidize fatty acids into the metabolic products carbon dioxide and water and heat (energy). Therefore there is a net oxidation of fuel with no use or storage of energy produced except in the form of heat (Cannon et al., Essays Biochem. 20:110, 1985).
The primary function of brown fat in mammals is to keep the mammal warm. Mammals have varying amounts and location of brown fat. Infants have larger areas of brown fat than adults.
Brown adipose tissue (BAT) is primarily concerned with maintenance of homeothermy through non-shivering thermogenesis (Cannon et al., Essays Biochem. 20:110, 1985). BAT has a high degree of vascularity, abundant mitochondria and high cytochrome concentrations in its mitochondria. Humans have brown fat located primarily in the back of the neck and subscapular regions. BAT is also located in the thorax, around the pericardium and sinoatrial node, along the aorta, around adrenal glands, and around sympathetic ganglia in the abdomen (Smith et al., Physiol. Rev. 49:330, 1969). With increasing age or obesity, brown fat becomes paler in color and more difficult to distinguish from white adipose tissue. White adipose tissue normally represents 15-25% of body weight and may reach up to 60% of body weight in massive obesity. Generally, BAT usually comprises less than 1% of total adult body weight.
Brown adipocytes (BA) contain multilocular lipid droplets and numerous mitochondria. BAT contains an abundant capillary plexus supplied by lobular arteries and drained by lobular veins. Further, BAT contains direct arteriovenous anastomoses, similar to those seen in liver (Nnodim et al., Am. J. Physiol. 182:283, 1988). BAT and blood vessels supplying BAT are innervated by sympathetic nerves as the only innervation from the autonomic nervous system (Cottle et al., Histochem. J. 17:1279, 1985).
White adipose tissue exports lipid to other tissues on demand. BAT oxidizes both endogenous and exogenous fatty acids. Therefore, BAT's physiologic role includes temperature regulation and possible maintenance of energy balance. When BAT was surgically removed from Osborne-Mendel or Zucker rats, the animals demonstrated increased body fat accumulation. Thus when functional BAT is reduced there is increased body fat accumulation. Decreased BA thermogenesis leads to altered energy balance and increased white fat deposition.
Brown fat cells uniquely contain and preferentially express an uncoupling protein called thermogenin, mitochondrial uncoupling protein or UCP. UCP uncouples the usual process of catabolism and storage of energy in the form of adenosine triphosphate (ATP) (Cannon et al., FEBS Lett. 150: 129, 1982). UCP is a 32 kD protein found in the inner membrane of BA mitochondria. UCP serves as a proton channel to decrease the transmembrane proton gradient which drives the electron transport system (Nicholls et al., Biochem. Biophys. Acta 549:1, 1979). This uncouples oxidative phosphorylation of ATP from oxidation of fat fuel substrates and increases the rate of oxidation. Norepinephrine activates uncoupling of oxidative phosphorylation mediated by UCP. Norepinephrine is thought to exert its effect through cAMP-activated hydrolysis of triacylglycerols to release free fatty acid. Free fatty acids mimic the effect of norepinephrine and are themselves potent uncouplers of oxidative phosphorylation. Purine nucleotides are considered negative modulators (Jezek et al., Fed Eur. Biochem. 243:1147, 1989).
UCP is expressed in response to external stimuli of cold -acclimation in hamsters and rats. Based upon gene expression in the mouse, four hours of cold stress to the whole animal led to a seven-fold increase in UCP mRNA. Administration of norepinephrine also increased UCP mRNA to a lesser extent. UCP expression can also be induced in preadipocytes grown in culture. UCP mRNA translation was maximally stimulated by Norepinephrine when the cells were in confluence and in the presence of insulin or thyroid hormones (e.g., T3, T4, etc.). See, for example, Rehnmark et al., Exp. Cell Res. 182:75, 1989 and Rehnmark et al., J. Biol. Chem. 265:16464, 1990.
UCP is a proton/anion transporter found in the inner mitochondrial membrane of brown adipocytes. The mouse, rat, hamster and human genes encoding for UCP have been isolated and sequenced. UCP gene expression is controlled at the level of transcription by signals that are activated after stimulation of brown adipocytes by norepinephrine. The UCP sequence is strongly homologous to several other ubiquitous mitochondrial carriers, such as ANT (adenine nucleotide translocator) and a mitochondrial phosphate carrier. Jacobsson et al. (J. Biol. Chem. 260:16250, 1985) reported an isolation of a murine cDNA clone derived from brown adipose tissue mRNA. The cDNA was cold-inducible. No sequence data were provided. Bouillard et al. (J. Biol. Chem. 261:1487, 1986) reported a complete cDNA sequence and corresponding protein sequence for rat UCP. The rat UCP gene has no N-terminal signal extension. Rat UCP has a calculated molecular weight of 33,042 daltons and 306 amino acid residues. Ridley et al. (Nucleic Acids Res. 14:4025, 1986) also reports the cDNA and corresponding protein sequence for rat UCP as a 306 amino acid polypeptide. Ridley et al. state that the rat UCP cDNA sequence is 91.5% homologous to hamster UCP on the protein level. Kozak et al. (J. Biol. Chem. 263:12274, 1988) report two cDNA sequences for murine UCP. The polypeptide has six .alpha.-helical hydrophobic transmembrane domains, each encoded by an exon.
In view of the function of BAT to oxidize fuel substrates and BA to be regulated by norepinephrine, free fatty acids and other metabolic regulators, there is a need in the art to design a system to utilize the unique metabolic properties of BA to tip the metabolic balance toward white adipose tissue catabolism while preserving muscle mass. The following invention was made to fulfill this need.